Protein phosphorylation/dephosphorylation in the inner membrane of potato tuber mitochondria

Dioxins invade the body mainly through the diet, and produce toxicity through the transformation of aryl hydrocarbon receptor (AhR). An inhibitor of the transformation should therefore protect against the toxicity and ideally be part of the diet. We examined flavonoids ubiquitously expressed in plant foods as one of the best candidates, and found that the subclasses flavones and flavonols suppressed antagonistically the transformation of AhR induced by 1 nM of 2,3,7,8-tetrachlorodibenzo-p-dioxin, without exhibiting agonistic effects that transform AhR. The antagonistic IC(50) values ranged from 0.14 to 10 microM, close to the physiological levels in human.     

Synovial fluid level of aggrecan ARGS fragments is a more sensitive marker of joint disease than glycosaminoglycan or aggrecan levels: a cross-sectional study

Introduction

The gene MICA encodes the protein major histocompatibility complex class I polypeptide-related sequence A. It is expressed in synovium of patients with rheumatoid arthritis (RA) and its implication in autoimmunity is discussed. We analyzed the association of genetic variants of MICA with susceptibility to RA.

Methods

Initially, 300 French Caucasian individuals belonging to 100 RA trio families were studied. An additional 100 independent RA trio families and a German Caucasian case-control cohort (90/182 individuals) were available for replication. As MICA is situated in proximity to known risk alleles of the HLA-DRB1 locus, our analysis accounted for linkage disequilibrium either by analyzing the subgroup consisting of parents not carrying HLA-DRB1 risk alleles with transmission disequilibrium test (TDT) or by implementing a regression model including all available data. Analysis included a microsatellite polymorphism (GCT)n and single-nucleotide polymorphisms (SNPs) rs3763288 and rs1051794.

Results

In contrast to the other investigated polymorphisms, the non-synonymously coding SNP MICA-250 (rs1051794, Lys196Glu) was strongly associated in the first family cohort (TDT: P = 0.014; regression model: odds ratio [OR] 0.46, 95% confidence interval [CI] 0.25 to 0.82, P = 0.007). Although the replication family sample showed only a trend, combined family data remained consistent with the hypothesis of MICA-250 association independent from shared epitope (SE) alleles (TDT: P = 0.027; regression model: OR 0.56, 95% CI 0.38 to 0.83, P = 0.003). We also replicated the protective association of MICA-250A within a German Caucasian cohort (OR 0.31, 95% CI 0.1 to 0.7, P = 0.005; regression model: OR 0.6, 95% CI 0.37 to 0.96, P = 0.032). We showed complete linkage disequilibrium of MICA-250 (D' = 1, r ² = 1) with the functional MICA variant rs1051792 (D' = 1, r ² = 1). As rs1051792 confers differential allelic affinity of MICA to the receptor NKG2D, this provides a possible functional explanation for the observed association.

Conclusions

We present evidence for linkage and association of MICA-250 (rs1051794) with RA independent of known HLA-DRB1 risk alleles, suggesting MICA as an RA susceptibility gene. However, more studies within other populations are necessary to prove the general relevance of this polymorphism for RA.     

A comparison of different purification methods of aggrecan fragments from human articular cartilage and synovial fluid

In the study of aggrecan fragmentation several methods to extract and purify aggrecan from cartilage and synovial fluid (SF) are used. This work compares and evaluates the effectiveness for purification of aggrecan of the most commonly used methods by the ratio of sulfated glycosaminoglycan (sGAG) to protein and by fragment analysis by Western blot. A novel method for purification of aggrecan fragments from SF by boiling (Boiled SF) is also presented.Of the sGAG extracted from cartilage by guanidinium, 66% was recovered by associative–dissociative cesium chloride density gradient centrifugation (A1D1–D3) with a 9 times higher ratio of sGAG to protein in the A1D1 fraction. Although less enriched in aggrecan, the Western blot aggrecan pattern of the guanidinium extracted sample resembled that of the combined patterns of the A1D1, A1D2 and A1D3 fractions.The recoveries of sGAG from SF purified by anion chromatography and Alcian blue precipitation were around 50%, while the recoveries were over 80% in the associative or dissociative density gradient fractions (A1 and D1) and Boiled SF. The purification compared to neat SF ranged from 9 times in boiled SF to 1800–1900 times in Alcian blue and D1 samples. To obtain reliable results when analyzing synovial fluid aggrecan fragments by Western blot, purification was necessary. The immuno-pattern of anion chromatography purified SF resembled the patterns of A1 and D1, while the pattern of Boiled SF resembled the D1 sample.This work suggests that aggrecan fragments extracted from cartilage by guanidinium need no further purification to be analyzed by Western blot, whereas aggrecan fragments in SF are best analyzed in the A1 and D1 fractions or in the Boiled SF sample.     

The presence of a short redox chain in the membrane of intact potato tuber peroxisomes and the association of malate dehydrogenase with the peroxisomal membrane

Peroxisomes and mitochondria were purified from potato tubers (Solanum tuberosum L. cv. Bintje) by differential centrifugation followed by separation on a continuous Percoll gradient containing 0.3 M sucrose in the lower half and 0.3 M mannitol in the upper half. The peroxisomes band at the bottom and the mitochondria in the middle of this type of gradient. Mitochondrial contamination of the peroxisomes was only 2% [as judged by cytochrome c oxidase (EC 1.3.9.1) activity]. Contamination by amyloplasts, plasma membrane and endoplasmic reticulum was also minimal. The peroxisomes were 80% intact as judged by malate dehydrogenase (MDH, NAD^?-dependent; EC 1.1.1.37) latency. The specific activity of NADH-ferricyanide reductase and NADH-Cyt c reductase was 0.22 and 0.051 μmol (mg protein)^?1 min^?1 in freshly isolated peroxisomes, respectively. The active site of the reductase appeared to be on the inner surface of the membrane. The peroxisomes also contained a b-type cytochrome. Frozen peroxisomes were subfractionated by osmotic rupture followed by centrifugation to separate the soluble proteins from the peroxisomal membrane. About half the MDH and 30% of the NADH-ferricyanide reductase activity was associated with the membrane but only 6% of the catalase (EC 1.11.1.6) activity. A further wash removed 75% of the residual catalase with only a small loss of MDH or NADH-ferricyanide reductase activity. MDH appears to be closely associated with the peroxisomal membrane. When the purified peroxisomal membrane was analyzed by SDS-PAGE followed by silver staining, prominent bands at 22, 40, 41, 48, 53 and 74 kDa were observed. After immunoblotting the purified peroxisomal membrane, a band at 53 kDa showed strong cross-reactivity with antibodies raised against NADH-ferricyanide reductase. Since the NADH-ferricyanide reductase activity in the peroxisomal membrane could be shown to be specific for the β-hydrogen of NADH, the activity could not be due to contamination by endoplasmic reticulum where the reductase is α-specific. We conclude that the peroxisomal membrane contains a short redox chain, consisting of a NADH-ferricyanide reductase and a b-type cytochrome, similar to that of e.g. the plasma membrane. The role of this redox chain has yet to be elucidated.     

MMP proteolysis of the human extracellular matrix protein aggrecan is mainly a process of normal turnover

Although it has been shown that aggrecanases are involved in aggrecan degradation, the role of MMP (matrix metalloproteinase) aggrecanolysis is less well studied. To investigate MMP proteolysis of human aggrecan, in the present study we used neoepitope antibodies against MMP cleavage sites and Western blot analysis to identify MMP-generated fragments in normal and OA (osteoarthritis/osteoarthritic) cartilage, and in normal, knee injury and OA and SF (synovial fluid) samples. MMP-3 in vitro digestion showed that aggrecan contains six MMP cleavage sites, in the IGD (interglobular domain), the KS (keratan sulfate) region, the border between the KS region and CS (chondroitin sulfate) region 1, the CS1 region, and the border between the CS2 and the G3 domain, and kinetic studies showed a specific order of digestion where the cleavage between CS2 and the G3 domain was the most preferred. In vivo studies showed that OA cartilage contained (per dry weight) 3.4-fold more MMP-generated FFGV fragments compared with normal cartilage, and although aggrecanase-generated SF-ARGS concentrations were increased 14-fold in OA and knee-injured patients compared with levels in knee-healthy reference subjects, the SF-FFGV concentrations did not notably change. The results of the present study suggest that MMPs are mainly involved in normal aggrecan turnover and might have a less-active role in aggrecan degradation during knee injury and OA.     

Synovial fluid level of aggrecan ARGS fragments is a more sensitive marker of joint disease than glycosaminoglycan or aggrecan levels: a cross-sectional study

Introduction  

Aggrecanase cleavage at the ³⁹²Glu-³⁹³Ala bond in the interglobular domain (IGD) of aggrecan, releasing N-terminal ³⁹³ARGS fragments, is an early key event in arthritis and joint injuries. Here, we use a quantitative immunoassay of aggrecan ARGS neoepitope fragments in human synovial fluid to determine if this cleavage-site specific method better identifies joint pathology than previously available less specific aggrecan assays.     

Association between synovial fluid levels of aggrecan ARGS fragments and radiographic progression in knee osteoarthritis

Introduction  

Aggrecanase cleavage at the³⁹²Glu-³⁹³Ala bond in the interglobular domain (IGD) of aggrecan, releasing N-terminal³⁹³ARGS fragments, is an early key event in arthritis and joint injuries. We determined whether synovial fluid (SF) levels of ARGS-aggrecan distinguish subjects with progressive radiographic knee osteoarthritis (ROA) from those with stable or no ROA.     

Phosphoproteins and Protein Kinase Activities Intrinsic to Inner Membranes of Potato Tuber Mitochondria

Inside-out submitochondrial particles (IO-SMP) were isolatedand purified from potato (Solanum tuberosum L. cv.) tubers.When these IO-SMP were incubated with [ ³²P]ATP more then 20proteins became labelled as a result of phosphorylation. The³²P incorporation was stimulated by the oxidising reagent ferricyanide.Except for a 17 kDa protein which was phosphorylated only inthe absence of divalent cations, the protein phosphorylationrequired Mg²⁺. The time for half-maximum ³²P incorporation was4 mm for the 22 kDa phospho-F₁ -subunit and 2 min for the 28kDa phospho-F₀ b-subunit of the proton-ATPase. The K_m for ATPfor the detected phosphoproteins was between 65 µM and110 µM. The pH optimum for protein phosphorylation ininner membranes was between pH 6 and 8, and for the F₁ -subunitand the F₀ b-subunit the pH optima were 6.5–8 and pH 8,respectively. A 37 kDa phosphoprotein was phosphorylated ona histidine residue while the remainder of the inner membraneproteins were phosphorylated on serine or threonine residues.Two autophosphorylated putative kinases were identified: oneat 16.5 kDa required divalent cations for autophosphorylation,while another at 30 kDa did not. A 110 kDa protein was labelledonly with [-³²P] suggesting adenylylation. ³ Present address; Novartis Seeds AB, Box 302, S-261 23 Landskrona,Sweden.     

Comparison of the Stereospecificity and Immunoreactivity of NADH-Ferricyanide Reductases in Plant Membranes

The substrate stereospecificity of NADH-ferricyanide reductase activities in the inner mitochondrial membrane and peroxisomal membrane of potato (Solanum tuberosum L.) tubers, spinach (Spinacea oleracea L.) leaf plasma membrane, and red beetroot (Beta vulgaris L.) tonoplast were all specific for the [beta]-hydrogen of NADH, whereas the reductases in wheat root (Triticum aestivum L.) endoplasmic reticulum and potato tuber outer mitochondrial membrane were both [alpha]-hydrogen specific. In all isolated membrane fractions one or several polypeptides with an apparent size of 45 to 55 kD cross-reacted with antibodies raised against a microsomal NADH-ferricyanide reductase on western blots.     

Purification of a serine and histidine phosphorylated mitochondrial nucleoside diphosphate kinase from Pisum sativum.

For the first time, to our knowledge, a nucleoside diphosphate kinase (NDPK) has been purified from plant mitochondria (Pisum sativum L.). In intact pea leaf mitochondria, a 17.4-kDa soluble protein was phosphorylated in the presence of EDTA when [gamma-32P]ATP was used as the phosphate donor. Cell fractionation demonstrated that the 17.4-kDa protein is a true mitochondrial protein, and the lack of accessibility to EDTA of the matrix compartment in intact mitochondria suggested it may have an intermembrane space localization. The 17.4-kDa protein was purified from mitochondrial soluble proteins using ATP-agarose and anion exchange chromatography. Amino-acid sequencing of two peptides, resulting from a trypsin digestion, revealed high similarity with the conserved catalytic phosphohistidine site and with the C-terminal of NDPKs. Acid and alkali treatments of [32P]-labelled pea mitochondrial NDPK indicated the presence of acid-stable as well as alkali-stable phosphogroups. Thin-layer chromatography experiments revealed serine as the acid-stable phosphogroup. The alkali-stable labelling probably reflects phosphorylation of the conserved catalytic histidine residue. In phosphorylation experiments, the purified pea mitochondrial NDPK was labelled more heavily on serine than histidine residues. Furthermore, kinetic studies showed a faster phosphorylation rate for serine compared to histidine. Both ATP and GTP could be used as phosphate donor for histidine as well as serine labelling of the pea mitochondrial NDPK.